Project Summary In skin epithelium, a population of progenitor cells distinctly capable of proliferation, self-renewal, and terminal differentiation into post-mitotic progeny, is responsible to sustain tissue homeostasis throughout life. Unlike proliferation and apoptosis, cell fate choices are currently assessed using qualitative or indirect assays. As a result, we lack full understanding of how progenitors balance proliferation with differentiation during skin development and homeostasis, or in growth disorders of the skin. Our long-term objective is to establish how epidermal progenitor cells balance growth in the presence of oncogenic mutations to maintain tissue homeostasis. To do so, we developed a novel assay to directly quantify rates of progenitor cell self-renewal and differentiation in epidermis during homeostatic and hyperproliferative growth. We will employ our assay in the mouse models engineered to express physiological levels of constitutively active Hras and Pik3ca in the skin. In addition, we will use our recently developed rapid gene- targeting method, to explore how specific effectors and substrates of RAS-PI3K signaling modify epidermal differentiation in vivo. This application aims to test the hypotheses that: 1.) Increased differentiation can balance mitogenic effects of oncogenic signaling to maintain tissue homeostasis; 2.) Ras effector Pik3ca initiates a molecular cascade that includes Akt and specific Akt substrates to regulate progenitor cell renewal and differentiation in skin epidermis; and 3.) Epidermal progenitor response to oncogene expression is heterogeneous and influenced by progenitor cell niche. The results of our research are expected to immediately uncover cellular and molecular principles that maintain skin homeostasis and functionality despite the abundance of growth-promoting mutations. These findings will be a critical step in development of pharmacological strategies to manipulate progenitor cell potential in the epidermis, to treat conditions marked by unrestrained tissue growth.